Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). GVHD decreases survival through excess non-relapse mortality (NRM), which, in the case of patients with hematologic malignancies, is not balanced by relapse protection through graft-versus-tumor (GVT) effects. During the development of GVHD, HCT donor T cells become activated against recipient host antigens, setting GVHD in motion, despite current immunosuppressive treatments. The central immunological tenet of transplantation is that both engraftment and tolerance can be achieved by manipulating T cell responses. However, immunosuppressive biologics targeting T cell co-stimulation through the CD28 pathway have proven unsatisfactory for controlling GVHD. A promising and long-standing alternate approach, based on the hypothesis that ?selective ligation of CTLA-4 attenuates in vivo T cell responses, prevents development of autoimmunity, and represents a novel immunotherapeutic approach for the induction and maintenance of peripheral tolerance? [1], remains untested because of the lack of any reagent, antibody-based or otherwise, that acts as a true agonist ligand of the inhibitory co-receptor CTLA-4 specifically, without CD28 cross-talk. The overall goal of this project is to finally test this hypothesis in the long-established canine model of acute GVHD (aGVHD) as the most stringent test of activated T cell downregulation, as a prelude to establishing this approach as a viable alternative for treating aGVHD as a consequence of HCT in humans. Overcoming the lack of a reagent, we have engineered a novel candidate immunotherapeutic based on a highly-multimerized form of canine CD80, HeptaCAN80. HeptaCAN80 specifically targets CTLA-4 without CD28 cross-reactivity. Multimerization achieves tight-binding through avidity, and efficiently cross-links CTLA-4, mimicking the native in vivo effects of physiological agonist ligands. HeptaCAN80 displays inherent cross-specificity for human CTLA-4 over human CD28, potentiating a direct path for translation to humans. The immediate goal of this R21 proposal is to complete the validation of HeptaCAN80, through in vitro biochemical and cell-base studies and in vivo toxicity and pharmacokinetic studies, in order to justify more extensive and expensive in vivo efficacy and mechanistic studies in follow-on projects in the canine aGVHD model. The significance of this work lies in: (1) evaluating an orthogonal, alternate approach for manipulating T cell responses using a unique reagent; and (2) ultimately making allogeneic HCT in human patients safer by dramatically reducing NRM. This approach would potentially benefit not only patients transplanted to treat advanced hematologic malignancies, but also patients transplanted for nonmalignant blood disorders such as aplastic anemia, immunodeficiency diseases, sickle cell anemia, and others.